1. Technical Field
The present invention relates to the microbiological industry, and specifically to a method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family which has been modified to attenuate expression of the cynTSX operon and the cynR gene.
2. Background Art
Conventionally, L-amino acids are industrially produced by fermentation methods utilizing strains of microorganisms obtained from natural sources, or mutants thereof. Typically, the microorganisms are modified to enhance production yields of L-amino acids.
Many techniques to enhance L-amino acid production yields have been reported, including transformation of microorganisms with recombinant DNA (see, for example, U.S. Pat. No. 4,278,765). Other techniques for enhancing production yields include increasing the activities of enzymes involved in amino acid biosynthesis and/or desensitizing the target enzymes of the feedback inhibition by the resulting L-amino acid (see, for example, WO 95/16042, or U.S. Pat. Nos. 4,346,170; 5,661,012 and 6,040,160).
Another way to enhance L-amino acid production yields is to attenuate expression of a gene, or several genes, involved in the degradation of the target L-amino acid, genes diverting the precursors of the target L-amino acid from the L-amino acid biosynthetic pathway, genes involved in the redistribution of carbon, nitrogen, and phosphate fluxes, and genes coding for toxins etc.
It is known that cyanate, a toxic compound, is the product of the spontaneous dissociation of urea in solution as well as of the decomposition of carbamoyl phosphate, an intermediate in the pyrimidine and arginine biosynthetic pathways. In Escherichia coli, the process of cyanate detoxication is mediated throught the cynTSX operon that enables E. coli to degrade and use cyanate as the sole nitrogen source. (Sung Y.-C. et. al., J. Biol. Chem.; 263(29):14769-14775 (1988)). The cynTSX operon which contains three genes: cynT, cynS, and cynX is transcribed as a polycistronic message. The cynTSX operon is inducible by cyanate and is under the control of the inducer gene cynR. This operon is also subject to catabolite repression by bicarbonate.
The product of the cynT gene of the cyn operon in Escherichia coli has been identified as a carbonic anhydrase. The cyn operon also includes the gene cynS, encoding the enzyme cyanase. Cyanase catalyzes the reaction of cyanate with bicarbonate to give ammonia and carbon dioxide. The carbonic anhydrase was isolated from an Escherichia coli strain overexpressing the cynT gene and characterized. The purified enzyme was shown to contain 1 Zn2+/subunit (24 kDa) and was found to behave as an oligomer in solution; the presence of bicarbonate resulted in partial dissociation of the oligomeric enzyme. The kinetic properties of the enzyme are similar to those of carbonic anhydrases from other species, including inhibition by sulfonamides and cyanate. The amino acid sequence shows a high degree of identity with the sequences of two plant carbonic anhydrases but not with animal and algal carbonic anhydrases. Since carbon dioxide formed in the bicarbonate-dependent decomposition of cyanate diffuses out of the cell faster than it would be hydrated to bicarbonate, the apparent function of the induced carbonic anhydrase is to catalyze hydration of carbon dioxide and thus prevent depletion of cellular bicarbonate (Guilloton M. B. et. al., J. Biol. Chem.; 267(6):3731-4 (1992)).
Cyanase CynS is composed of 8-10 identical subunits (Mr=17,008) with significant amounts of α-helix and β-sheet structures, which catalytic activity links to the structural integrity of the oligomer (Little R. M. et a., J. Biol. Chem.; 262(21):10120-10126 (1987)).
The cynX gene encodes hydrophobic protein CynX with undetermined function. Based exclusively on degrees of sequence similarity, CynX has been determined to be a member of the major facilitator superfamily (MFS), which is one of the two largest families of membrane transporters and is present ubiquitously in bacteria, archaea, and eukarya, as a putative cyanate transporter (Pao S. S., Microbiol. And Mol. Biol. Rev.; 62(1):1-34 (1998)).
The physiological role of cynT, cynS and cynX was investigated by construction of chromosomal mutants in which these genes were rendered inactive. The delta cynT chromosomal mutant expressed an active cyanase but no active carbonic anhydrase. In contrast to the wild-type strain, the growth of the delta cynT strain was inhibited by cyanate, and the mutant strain was unable to degrade cyanate and therefore could not use cyanate as the sole nitrogen source when grown at a partial CO2 pressures (pCO2) of 0.03% (air). At a high pCO2 (3%), however, the delta cynT strain behaved like the wild-type strain; it was significantly less sensitive to the toxic effects of cyanate and could degrade cyanate and use cyanate as the sole nitrogen source for growth. These results are consistent with the proposed function for carbonic anhydrase (Guilloton M. B. et. al., J. Bacteriol.; 175(5):1443-51 (1993)).
It was found that the carbonic anhydrase YadF is essential for cell growth in the absence of another carbonic anhydrase, CynT, in Escherichia coli. However, mutant strains lacking both of them grew at high CO2 concentrations (5%), where non-enzymatic mechanisms generate HCO3−. This suggests that these carbonic anhydrases are essential because they maintain HCO3− levels at ambient CO2 concentrations (Hashimoto M., and Kato J., Biosci. Biotechnol. Biochem.; 67(4):919-22 (2003)).
CynT is the single E. coli Can (previously YadF) paralog. It can, when induced with azide, replace Can (Merlin C., et. al., J. Bacteriol.; 185(21):6415-24 (2003)).
Expression of the cynTSX operon is controlled by the CynR protein encoded by the cynR gene and transcribed in the direction opposite to that of the cyn operon (Lamblin A.-F. J., et. al., J. Bacteriol.; 175(21):7990-7999 (1993)). Analysis of the β-galactosidase transcriptional fusion suggest that CynR is a dual transcriptional regulator that belongs to the LysR family and is negatively autoregulated independently of cyanate (Sung Y.-C., et al., J. Bacteriol.; 174(11):3645-3650 (1992)). CynR is 32,000 Mr protein that recognizes a 136-bp DNA fragment encompassing the cynR-cynTSX intergenic region. Circular permutation assays indicated that CynR induces bending of the DNA upon binding and that this bending decreases in the presence of cyanate (Lamblin A.-F. J., et. al., J. Bacteriol.; 176(21):6613-6622 (1994)).
But currently, there have been no reports of attenuating expression of the cynTSX operon and the cynR gene for the purpose of producing L-amino acids.